For in vitro DNA packaging assays, each 20 μl of reaction mixture contained purified T4 heads (∼2 × 1010 particles), purified full-length gp17 (∼3 μM), and linearized DNA in packaging buffer [30 mM tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl2, and 1 mM adenosine 5′-triphosphate (ATP)]. The mixture was incubated at 37°C for 30 min, followed by benzonase nuclease addition and incubation at 37°C for 20 min to remove excess unpackaged DNA. The encapsidated nuclease-resistant DNA was released by treatment with 50 mM EDTA, proteinase K (0.5 μg/μl; Thermo Fisher Scientific, MA), and 0.2% SDS for 30 min at 65°C. The packaged DNA was analyzed by 1% (w/v) agarose gel electrophoresis followed by staining with ethidium bromide, and the amount of packaged DNA was quantified using Quantity One software (Bio-Rad, CA). The packaging efficiency was defined as the number of DNA molecules packaged per T4.

In vitro protein display on the T4 head was assessed by the co-sedimentation described previously (8). Briefly, after encapsidating linearized DNA as described above, T4 heads were incubated with Soc- and/or Hoc-fused proteins at 4°C for 45 min. The mixtures were sedimented by centrifugation at 30,000g for 45 min, and unbound proteins in the supernatants were removed. After washing twice with PBS, the pellets were incubated at 4°C overnight and then resuspended in PBS for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis or Opti-MEM for transduction. After Coomassie Blue R-250 (Bio-Rad, CA) staining and destaining, the protein bands on SDS-PAGE gels were scanned and quantified by laser densitometry (Personal Densitometer SI; GE Healthcare, IL). The densities of the Hoc, Soc, and gp23 bands were determined for each lane separately, and the copy numbers of bound Hoc or Soc fusion molecules per capsid were calculated using gp23 as the internal control (930 copies per capsid).

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